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Hepatitis B virus multi-locus genotypic resistance mutation detection method

A virus, whole gene technology, applied in the field of hepatitis B virus multi-locus gene resistance mutation detection, can solve the problems of increasing the chance of PCR amplification product contamination, difficult multi-locus gene mutation detection, unsuitable simultaneous detection, etc. To achieve the effect of improving specificity, high sensitivity and eliminating influence

Inactive Publication Date: 2010-04-28
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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AI Technical Summary

Problems solved by technology

The two rounds of traditional nested PCR reactions are carried out in two tubes respectively, resulting in the following disadvantages: first, it increases the chance of contamination of PCR amplification products; second, it is not suitable for simultaneous detection of samples with large differences in viral load; third, it increases testing cost
[0014] With the increase of the types of anti-HBV nucleoside (acid) drugs, the number and mutation forms of HBV drug-resistant mutations are also increasing, and the real-time fluorescent PCR method commonly used in clinical detection of YMDD mutations is difficult to detect multi-site gene mutations at the same time
[0015] However, there is currently no mature product for detecting HBV multi-site drug-resistant mutations on the market. Therefore, the development of PCR products with high sensitivity, wide detection range of viral load, strong specificity, simplicity, convenience, and low price that are easy for patients to accept directly Sequencing method to detect HBV drug resistance mutation is imminent

Method used

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  • Hepatitis B virus multi-locus genotypic resistance mutation detection method

Examples

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Embodiment 1

[0044] Embodiment 1 Single-tube nested PCR technology amplifies the whole gene sequence of hepatitis B virus reverse transcriptase

[0045] Design of amplification primers: 729 full-length HBV genome sequences were selected from GenBank, including 209 sequences of Chinese-specific genotypes B and C, and the conserved sequences of the upstream and downstream of the HBV reverse transcriptase gene were compared and analyzed with NTI software. Two rounds of PCR primers were designed as follows: the PCR product was 1225bp, including the full-length 1032bp HBV reverse transcriptase region (nucleotides 130-1161). The second-round primers also serve as sequencing primers.

[0046] SEQ ID NO: 1-4 respectively correspond to the upstream and downstream of the outer primer and the upstream and downstream of the inner primer, see Table 1.

[0047] Table 1 Internal and external primers and positions designed to amplify the whole gene of HBV reverse transcriptase

[0048]

[0049] The e...

Embodiment 2

[0086] Example 2 Single-tube nested PCR gene amplification method to detect hepatitis B virus drug-resistant mutation sites

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Abstract

The invention discloses a hepatitis B virus multi-locus genotypic resistance mutation detection method. A single tube nest type polymerase gene amplification method for detecting reverse transcriptase full gene sequence of virus includes that nest type PCR amplification is carried out in a single tube for twice, products of a first group PCR reaction are all used as templates of a second group PCR reaction by adjusting concentration of internal and external primers, and finally nest type PCR reaction is completed in the same single tube. The invention ensures specificity and economy of detection while obviously improving detection sensibility and has the detection range over a hundred million difference viral load.

Description

Technical field: [0001] The invention relates to a method for detecting drug-resistant mutations of hepatitis B virus multi-site genes, in particular to a single-tube nested polymerase chain reaction (PCR) amplification method for detecting hepatitis B virus (HBV) reverse transcriptase full gene sequence. Background technique: [0002] Nucleoside (acid) drugs are the main drugs for antiviral treatment of hepatitis B. Currently, the targets of anti-HBV nucleoside (acid) drugs in clinical use are all HBV reverse transcriptase (reverse transcriptase, RT), and long-term use will produce RT. For the problem of genetic drug resistance mutations, if drug resistance mutations are detected before medication, it can guide clinical rational drug use and avoid the impact of primary drug resistance mutations; if drug resistance is detected at the genetic level or early in virus breakthrough, clinical intervention can be carried out as soon as possible. Disease rebound can be avoided; ev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 徐东平刘妍钟彦伟李晓东戴久增王琳李乐
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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